Project Summary A major breakthrough in neural development was the identification of attractive and repulsive molecules that guide neurons and their processes to their targets. Today, textbook models emphasize the importance of molecular gradients that act over long-range to steer neurons, epitomized by Netrin-1 (Ntn-1) and its receptor Deleted in colorectal cancer (DCC). Recently, this idea has come into question with the discovery that some of Ntn1's classic long-range effects may be better explained by its short-range, permissive functions. Additionally, the classic model does not adequately explain the remarkable reliability of guidance in vivo, where axons navigate through a complex and changing environment. Structural biological observations suggest a provocative new mechanism that reconciles conflicting observations and could enable greater fidelity than what is possible with a simple gradient. Our long-term goal is to define the molecular mechanism by which Ntn1 achieves both short and long-range functions, even via the same DCC receptor. The overall objective of this exploratory project is to create two new mouse lines so we can test structural predictions in vivo and determine whether a long- term, deeper investigation is warranted. Traditional models for chemoattraction are based largely on the activities of individual ligands and receptors and do not account for the ample cross-talk that also occurs. For example, Ntn-1 binds directly to the secreted protein Draxin, and both ligands bind to DCC, but through independent sites. Further, there are two isoforms of DCC, one short (DCCshort) and one long (DCClong), whose expression changes over development. By solving the structures of Ntn1 and Draxin bound to each other and to DCC, our collaborators determined that Ntn1 can participate in two distinct complexes: a Draxin-mediated 1:2 Ntn1/DCC complex and a DCClong- facilitated 1:1 Ntn1/DCC complex that triggers clustering and hence signaling. The realization that Ntn1's activity might be shaped both by the presence of Draxin and the nature of DCC offers a satisfying explanation for how Ntn1 can act permissively in some contexts and instructively in others, thereby steering cells and axons through complex environments with greater accuracy. We hypothesize that Ntn1 signals through different complexes to mediate short-range, adhesive interactions versus long-range guidance in response to a gradient. To begin to test this hypothesis, we will pursue the following three specific aims: 1) to create a mouse strain deficient for Draxin-DCC binding; 2) to create a mouse strain that cannot produce DCClong; and 3) to test whether predicted changes in the nature of the Ntn1/DCC complex differentially affect Ntn1's ability to act permissively or instructively in vivo. These studies will position us to define a conceptually novel mechanism for guidance and establish a new paradigm for Ntn1 signaling, with broad implications for neuroscience and cancer biology.